A. Field of the Invention
The present invention relates generally to the fields of protein chemistry, biochemistry, organic chemistry and molecular biology. More particularly, it concerns a novel screen for identifying peptides having target binding affinity.
B. Description of Related Art
With the end of the human genome project in sight, the next great challenge in human biology will be to deduce the function of the thousands of new gene products identified by the sequencing effort. Perhaps the most direct way to take advantage of knowing the sequences of these proteins would be to design compounds capable of binding specific epitopes in a factor of interest. These binding agents could then be used for a variety of purposes, including affinity purification of the protein or the manipulation of its post-translational modification (see below).
Unfortunately, peptides, or peptide epitopes in proteins, are difficult targets for molecular recognition studies in aqueous solution. An unstructured peptide, or even one with a well-defined secondary structure, does not present to a prospective ligand the kind of molecular crevices and canyons present in a typical globular protein or in a double helical nucleic acid, which shield interacting groups from competition by solvent water. Indeed, looking at the prior art, it was not clear that it would be possible to form stable, highly specific, non-covalent complexes between peptide targets and small molecules in water. Leucine zipper domains are the smallest known naturally occurring motif that supports sequence-specific interactions, and these moieties are comprised of 30 or more amino acids (Landschulz et al., 1990; O'Shea et al., 1992) there are no natural examples of short peptide sequences that are independently capable of mediating protein-protein interactions.
There has been considerable interest in the organic chemistry community in developing synthetic peptide receptors. While some elegant chemistry has been performed, the receptors that have evolved from these efforts are far from being practically useful to biologists. Still and co-workers searched synthetic combinatorial libraries of cyclic amides for molecules able to bind tripeptides and incorporated these into fluorescence-based sensor systems (Burger and Still, 1997; Chen et al., 1998; Cheng et al., 1996; Shao and Still, 1996; Still, 1996). Complexes with KDS of 20–40 μM were obtained, but these receptors and the target peptides only associated in chloroform. The literature on receptor-peptide binding in water is even less developed. For example, Hossain & Schneider (Hossain and Schneider, 1998) describe tripartite receptors for di- and tripeptides comprised of a crown ether and a quaternary amine separated by a hydrophobic spacer. These bind hydrophobic di- and tripeptides in water with KDs in the mM to μM range, and very modest sequence specificity. These receptors were designed specifically to utilize the charged N- and C-terminal ends of a small peptide in binding, and so would not be applicable to the recognition of peptide epitopes in proteins.
The only report in the literature of peptide-like epitope-binding molecules that function in water is that of Nestler and co-workers at Cold Spring Harbor (Dong et al., 1999). These workers employed synthetic, bead-bound libraries of “forcep” molecules as potential epitope receptors. These forceps were comprised of multiple copies of a single peptide or peptide-like molecule displayed on either a stiff or floppy molecular scaffold. Forceps that bind an epitope from Ras were isolated and shown to be able to inhibit the famesylation of Ras when present at high concentrations. However, the Nestler study was restricted to these homo-oligomeric forceps, which bind their target weakly. It was not demonstrated that this is a system of general utility, nor was it demonstrated that simple epitope-binding peptides lacking an elaborate superstructure could be isolated. Finally, the method employed by Nestler and co-workers requires a synthetic target molecule.
At present, the only class of molecules generally useful for peptide recognition in water is antibodies. Antibodies are, of course, proteins; not low molecular weight compounds. They are relatively fragile compared to small molecules. Using classical methods, they are tedious and expensive to obtain, particularly in large quantities, although advances in the construction of single chain antibody libraries on phage (Griffiths and Duncan, 1998; Rader and Barbas, 1997) promise to speed up this process. Finally, antibodies are not easily rendered cell-permeable.
Another major issue with antibodies is one of scale. For example, immunoaffinity purification of multi-protein complexes containing an epitope-tagged protein is an important tool in probing the biochemistry of splicing, transcription, DNA replication and many other critical cellular processes. However, they are extremely expensive to carry out on a large scale since the commercially available monoclonal antibodies used for these studies sell for more than $100 per milligram. Furthermore, the antibody-epitope interaction is generally so tight, that yields of only 1–10% are realized when one attempts to elute the tagged complex from the column with synthetic epitope peptide. This also makes reuse of the resin very difficult. This technology is good enough to provide quantities sufficient for identification of the component polypeptides by microsequencing techniques, but it is difficult to prepare enough material for extensive biochemical analysis, let alone structural studies. Thus, a major goal in the proteomics area is to develop synthetic epitope receptors that function in water and bind their targets selectively with modest to very high affinities.
In order to achieve all the goals necessary for such projections, the following attributes are required of the desired affinity reagent. First, the epitope-binding molecule (EBM) must bind to a given epitope (sequence of about 5–15 amino acids) in a protein of interest with very high specificity. Furthermore, it is critical that the inventor is able to choose the epitope. Screens carried out using intact recombinant protein targets almost invariably identify molecules that recognize natural interaction sites, probably because these represent the most “bindable” surface of a protein (Fairbrother et al., 1998; Zhu et al., 2000). However, the inventor wishes to isolate native complexes where these interaction surfaces will generally be occupied, so the affinity reagent must recognize some other surface of the protein target.
Second, EBMs of both moderate and high affinity must be available. High affinity EBMs will be of utility in the construction of biosensors (Kodadek, 2001) and in other applications as antibody replacements. However, for the affinity purification of a protein and its associated factors, EBMs of modest affinity will be much more useful, since the target protein can be eluted much more efficiently from a modest affinity EBM under native conditions than from a tight-binding antibody. Based on data that will be discussed below, the inventors believe that for affinity chromatography applications, a KD between 10−4 M and 10−7 M would be ideal (Zhang et al., 2000). For biosensor applications, EBM-protein complexes with KDs of 10−9 M or below are desired.
Third, the EBM must be a relatively small molecule that can be synthesized in at least milligram, and preferably gram, quantities. Fourth, the screen used to identify the EBM must be relatively rapid and convenient, so as to be capable of supporting high-throughput. Thus, it is clear that there remains a considerable need in the field for a reagent with all of foregoing properties.